Characterization Of Three Chimeric ?-Amylases (AmyP) By Fusion Of Exogenous Starch Binding Domains (SBD)

a-amylase is an important industrial enzyme,widely used in food,chemical,brewing and other industries.?-amylase AmyP,which has the ability to degrade starch,is derived from the marine metagenomic library.It can not only degrade many kinds of raw starch,but also has a preference for raw rice starch.AmyP has a starch binding domain SBD,the deletion of the SBD will not decrease the ability of AmyP to degrade starch reduced dramatically.In order to improve the bility of AmyP to degradation starch,we replaced the SBD of AmyP,Selection the SBD of a-amylase from Cryptococcus sp.S-2,Thermobifda fusca NTU22 ? Clostridium butyricum T-7 fusion with AmyP?SBD.The recombinant enzyme was expressed in E.coli and obtained three fusion enzymes AmyP-Cr?AmyP-Th and AmyP-Cl.The fusion enzyme AmyP-Cr is fusion of AmyP?SBD and SBD of a-amylase of Cryptococcus sp.S-2,which has the best enzymatic properties in the the three fusion enzyme.The specific activity of AmyP-Cr to raw rice starch was 327.9�31.8 U/mg,which was 1.7 times of wild type AmyP.The highest degradation rate of AmyP-Cr was 46.6%,which was about 1.8 times of wild type AmyP in 4h.The highest binding ratio of AmyP-Cr to raw rice starch was 0.113 uM/g,which was about 5.1 times of AmyP.Moreover,the thermal stability of AmyP-Cr was improved,and the half-life of AmyP-Cr at 40? was above 12 h,which was 5 times of that of wild type AmyP.These data indicated that improve of the degradation ability of ability of AmyP-Cr to raw rice starch,was due to the SBD of Cryptococcus sp.S-2 could improve the dsorbability and thermal stability of ezyme to the raw rice substrate at the same time.AmyP-Cr is a new rice starch degrading enzyme with excellen properties.The fusion enzyme AmyP-Th is fusion of AmyP?SBD and SBD of a-amylase of Thermobifida fusca NTU22.The specific activity of AmyP-Th to raw rice starch was 254.2�26.5 U/mg,which was 1.3 times of wild type AmyP.The highest degradation rate of AmyP-Th was 39.6%in 4h,although that was 1.5 times higher than wild type AmyP,but that was slightly lower than AmyP-Cr.The fusion enzyme AmyP-Cl is fusion of AmyP?SBD and SBD of a-amylase of Clostridium butyricum T-7.The specific activity of AmyP-Cl to raw rice starch was 468.6�43.5 U/mg,which Was 2.4 times of wild type AmyP.Although the activity of AmyP-Cl was highest in the three fusion proteins,but the highest degradation rate of AmyP-Cl towards rice starch was 41.6%in 4h,slightly lower than AmyP-Cr.In addition,the thermal stability of AmyP-Cl was not significantly improved.